Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug

Abstract

Standard literature procedures for the chemical synthesis of L-threose nucleosides generally employ L-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either L-arabitol or L-diethyl tartrate, both affording 2-O-methyl-L-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2′-deoxy-α-L-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.

Practical and General Alcohol Deoxygenation Protocol

“…Deoxygenation of electron deficient and hindered substrate giving product 10 highlights the unique utility of this method; this substructure is not amenable to alternative deoxygenation strategies based on carbocation formation, substitution, or elimination. A variety of unsaturated heterocycles are tolerated in this process, such as furan (11), pyrazole (12), triazole, (13), purine (14), and imidazole (15). Selective deoxygenation of a benzoate substrate bearing an aryl chloride yielding 15 is of particular note given that previous reports generating CO 2 *À from formate have focused on aryl radical generation from aryl halides.…”

“…[8] Deoxygenation processes are typically deployed in two distinct contexts: (1) removal of oxygen-based functional groups after they enable robust CÀ C and CÀ X bond forming reactions (e.g., carbonyl reactivity, Figure 1B) [9][10][11][12] and (2) selective removal of specific oxygen atoms from complex polyoxygenated feedstocks, such as carbohydrates, to access chiral building blocks (Figure 1C). [13][14][15] Unfortunately, the current standard protocol to remove alcohols, the Barton-McCombie deoxygenation, relies on stoichiometric trialkyl tin hydride and thiocarbonyl-based activating groups (Figure 1D). [16] While Barton-McCombie variants designed to reduce or eliminate trialkyl tin reagents have been developed, these approaches remain limited in scope and still require unstable thiocarbonyl activating groups, which can also be difficult to install.…”

Crystallographic analysis of engineered polymerases synthesizing phosphonomethylthreosyl nucleic acid

“…The ability for Kod-RI to synthesize TNA has been extensively studied by X-ray crystallography and structures now exist for each of the major intermediates in the TNA synthesis pathway, including the apo, binary, open and closed ternary complexes of the pre-catalytic state as well as the translocated post-catalytic product ( 20 ). Recognizing that pTNA is a close structural analogue of TNA (Figure 1A ), we wondered whether Kod-RI might also facilitate pTNA synthesis by recognizing chemically prepared ptNDP nucleotides ( 17 , 37 ), the diphosphate monophosphonate building blocks of pTNA, in a template-dependent primer extension reaction (Figure 1A ). Our findings indicate that Kod-RI is capable of synthesizing trace amounts of full-length pTNA after 24 h of incubation at 55°C (Figure 1B ).…”